Parent-assisted anaesthesia.
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Anesthesia, Inhalation↗
Biomedical subjects
T Gallagher
Publications and source records attributed to T Gallagher.
Subtilisin BPN' at 1.6 A resolution: analysis for discrete disorder and comparison of crystal forms.
The three-dimensional structure of the serine protease subtilisin BPN' (SBT) has been refined at 1.6 A resolution in space group C2 to a final R value of 0.17. 17 regions of discrete disorder have been identified and analyzed. Two of these are dual-conformation peptide units; the remainder involve alternate rotamers of side chains either alone or in small clusters. The structure is compared with previously reported high-resolution models of SBT in two other space groups, P2(1)2(1)2(1) and P2(1). Apart from the surface, there are no significant variations in structure among the three crystal forms. Structural variations observed at the protein surface occur predominantly in regions of protein-protein contact. The crystal packing arrangements in the three space groups are compared.
Journal Article↗
The prosegment-subtilisin BPN' complex: crystal structure of a specific 'foldase'.
BACKGROUND: The folding of the bacterial protease subtilisin BPN' (SBT) is dependent on its 77-residue prosegment, which is then autocatalytically removed to give the mature enzyme. Mature subtilisin represents a class of proteins that lacks an efficient folding pathway. Refolding of mature SBT is extremely slow unless catalyzed by the independently expressed prosegment, leading to a bimolecular complex. RESULTS: We report the crystal structure at 2.0 A resolution of the prosegment-SBT complex and consider its implications for prosubtilisin BPN' maturation and folding catalysis. The prosegment forms a compact domain that binds SBT through an extensive interface involving the enzyme's two parallel surface helices (residues 104-116 and 133-144), supplying negatively charged caps to the N termini of these helices. The prosegment C terminus binds in the enzyme active site in a product-like manner, with Tyr77 in the P1 binding pocket. CONCLUSIONS: The structure of the complex supports a unimolecular mechanism for prosubtilisin cleavage, involving a 25 A rearrangement of the SBT N terminus in a late folding step. A mechanism of folding catalysis in which the two helices and their connecting beta strand form a prosegment-stabilized folding nucleus is proposed. While this putative nucleus is stabilized by prosegment binding, the N-terminal and C-terminal subdomains of SBT could fold by propagation.
Crystallography, X-Ray↗
Catalysis of a protein folding reaction: mechanistic implications of the 2.0 A structure of the subtilisin-prodomain complex.
Biosynthesis of subtilisin is dependent on a 77 amino acid, N-terminal prodomain, which is autocatalytically processed to create the mature form of the enzyme [Ikemura, H., Takagi, H., & Inouye, M. (1987) J. Biol. Chem. 262, 7859-7864]. In order to better understand the role of the prodomain in subtilisin folding, we have determined the structure of the processed complex between the prodomain and subtilisin Sbt-70, a mutant engineered for facilitated folding. The prodomain is largely unstructured by itself but folds into a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helices when complexed with subtilisin. The Ka of the complex is 2 x 10(8) M-1 at 25 degrees C. The prodomain binds on subtilisin's two parallel surface alpha-helices and supplies caps to the N-termini of the two helices. The C-terminal strand of the prodomain binds in the subtilisin substrate binding cleft. While Sbt-70 is capable of independent folding, the prodomain accelerates the process by a factor of > 10(7) M-1 of prodomain in 30 mM Tris-HCl, pH 7.5, at 25 degrees C. X-ray structures of the mutant subtilisin folded in vitro either with or without the prodomain are compared and show that the identical folded state is achieved in either case. A model of the folding reaction of Sbt-70 and the prodomain is described as the following equilibria: P + Su<-->Pf--SI<-->Pf--Sf, where Su and P are Sbt-70 and prodomain, respectively, which are largely unstructured at the start of the reaction, Pf--SI is a collision complex of a partially folded Sbt-70 and folded prodomain, and Pf--Sf is the complex of folded Sbt-70 and prodomain.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Errors in administration of intravenous drugs.
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Humans↗
Anatoxin-a is a potent agonist of the nicotinic acetylcholine receptor of bovine adrenal chromaffin cells.
(+)-Anatoxin-a is a neurotoxic alkaloid produced by the cyanobacterium Anabaena flos-aquae. In this study synthetic (+/-)-anatoxin-a was tested on isolated bovine adrenal chromaffin cells to determine its ability to evoke secretion of endogenous catecholamines through neuronal-type nicotinic receptor activation. Anatoxin-a was found to act as a potent agonist of the secretory response of chromaffin cells with an EC50 of 1-2 microM, compared with an EC50 of 4-5 microM for nicotine. The cells responded to anatoxin-a and nicotine with bell-shaped concentration-response curves consistent with desensitisation at concentrations of anatoxin-a greater than 5 microM and of nicotine greater than 20 microM. The secretion of catecholamines stimulated by anatoxin-a was completely inhibited in a non-competitive manner by the nicotinic antagonist mecamylamine with an IC50 of 0.4-0.5 microM. In the presence of depolarising concentrations of K+ (15 or 50 mM), anatoxin-a increased the secretion of catecholamines in a concentration-dependent manner up to the same maximum as that achieved by anatoxin-a alone. It is concluded that anatoxin-a acts as a potent and selective nicotinic agonist, capable of evoking secretion of endogenous catecholamines from chromaffin cells via their neuronal-type nicotinic receptor.
Animals↗
Anatoxin-a-evoked [3H]dopamine release from rat striatal synaptosomes.
Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. Using rat striatal synaptosomes loaded with [3H]dopamine, we have characterized the action of the selective nicotinic agonist, (+/-)anatoxin-a, with respect to [3H]dopamine release, in order to explore the mechanisms coupling nicotinic receptor activation to exocytosis. Anatoxin-a evoked [3H]dopamine release in a concentration-dependent and mecamylamine-sensitive manner, EC50 = 0.11 microM. The maximum [3H]dopamine release elicited by anatoxin-a was only 20% of the maximum elicited by KCl depolarization; there was no additivity between anatoxin-a and sub-maximal concentrations of KCl. Both agents stimulated Ca(2+)-dependent release that was equally sensitive to inhibition by 200 microM Cd2+. This result suggests that anatoxin-a-stimulated exocytosis is mediated by Ca2+ influx via voltage-sensitive Ca2+ channels, with little contribution from Ca2+ entering directly through the nicotinic receptor channel. This view is supported by the abolition of anatoxin-a-evoked [3H]dopamine release in Na(+)-depleted medium. A partial (40%) inhibition by tetrodotoxin was observed. These data suggest that activation of presynaptic nicotinic acetylcholine receptors by anatoxin-a results in an influx of Na+, producing sufficient local depolarization to open voltage-sensitive Ca2+ and Na+ channels. The latter may then amplify the response, activating further Ca2+ channels. The particular voltage-sensitive Ca2+ channels involved remain to be determined.
Animals↗
Stress and coping strategies in community psychiatric nurses: a Q-methodological study.
With the development of the concept of community care there has been a significant expansion of the community psychiatric nurse (CPN) profession. The present study attempts to examine which aspects of their work CPNs currently find stressful. The study also examines the various strategies which CPNs feel to be useful in attempting to cope with such occupational stress. Forty-four CPNs in four health districts participated in this Q-methodological study which provided the opportunity for CPNs to construct their own concepts of stressors and coping strategies. The results obtained indicated that CPNs identified nine distinct areas of stress within their work, along with 12 distinct coping strategies which they considered useful in attempting to deal with such stress. The implications of these findings are discussed.
Adaptation, Psychological↗
Purification of type 1 protein (serine/threonine) phosphatases by microcystin-Sepharose affinity chromatography.
A microcystin (MC)-Sepharose column was prepared by addition of 2-aminoethanethiol to the alpha, beta-unsaturated carbonyl of the N-methyldehydroalanine residue of MC-LR, followed by reaction of the introduced amino group with N-hydroxysuccinimide-activated CH-Sepharose. The MC-Sepharose bound protein phosphatase-1 (PP1) with high capacity and purified human PP1 gamma in one step from E. coli extracts. It was also used to purify forms of PP1 bound to myofibrils from skeletal muscle. Two of these comprised PP1 complexed to N-terminal fragments of the M-subunit which enhance its myosin phosphatase activity, while the third comprised PP1 and an N-terminal fragment of the glycogen-binding (G)-subunit.
Animals↗
Two crystal structures of the B1 immunoglobulin-binding domain of streptococcal protein G and comparison with NMR.
The structure of the 56-residue B1 immunoglobulin-binding domain from streptococcal protein G has been determined in two different crystal forms. The crystal structures were deduced by molecular replacement, based on the structure of the B2 domain (Brookhaven accession code 1PGX). Final R values are 0.174 and 0.198 for orthorhombic and trigonal forms, for diffraction data from 6.0 to 2.07 A and from 6 to 1.92 A, respectively. The orthorhombic crystals have an unusually high packing density for protein crystals, with Vm = 1.66 and a solvent content of 26%. The protein structure is found to be very similar (rms deviation 0.25 A for 56 C alpha's) in the two crystal forms, with an efficiently packed hydrophobic core between a four-stranded beta-sheet and a four-turn alpha-helix. The B1 domain has the same fold and general structure as the B2 domain (rms deviations 0.36 and 0.39 A), despite the six residue differences between them. The crystallographic models differ from NMR-derived models in several local regions, primarily in the loop involving residues 46-51; other significant variations are observed in the helix and in the structure of bound water. The primary crystal contact is the same in both crystal forms, involving both sheet edges to form extended beta-sheets throughout the crystals.
Amino Acid Sequence↗
An engineered disulfide cross-link accelerates the refolding rate of calcium-free subtilisin by 850-fold.
The mature form of subtilisin is an unusual example of a monomeric protein with a high kinetic barrier to folding and unfolding. Using site-directed mutagenesis of subtilisin BPN', we are attempting to determine the physical and energetic nature of the kinetic barrier. The high-affinity calcium-binding site A has been shown to create a large enthalpic barrier to unfolding. Removing the calcium-binding site A from subtilisin by deleting amino acids 75-83 greatly accelerates both unfolding and refolding reactions. Here a disulfide cross-link is introduced between residues 22 and 87 in delta 75-83 subtilisin. This was done to probe the conformational entropy of the transition state for folding. The 1.8-A X-ray structure of this mutant and the effects of the cross-link on the kinetics of unfolding and refolding are reported. Consistent with an expected loss of entropy of the unfolded protein due to the cross-link, the disulfide accelerates folding relative to the uncross-linked form. The magnitude of the acceleration of folding rate (700-850-fold at 25 degrees C) indicates that residues 22 and 87 are ordered in the transition state such that the disulfide does not affect its total entropy. Although early organization of structure around amino acids 22 and 87 greatly accelerates folding, we do not know whether the early folding of this region is a highly populated folding pathway in the absence of the cross-link. The slow step in the delta 75-83 subtilisin folding reaction may be forming initial structures capable of propagating the folding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Binding Sites↗
Refined structure of the pyruvoyl-dependent histidine decarboxylase from Lactobacillus 30a.
The crystal structure of the pyruvoyl-dependent histidine decarboxylase from Lactobacillus 30a has been refined to an R-value of 0.15 (for the 5.0 to 2.5 A resolution shell) and 0.17 (for the 10.0 to 2.5 A resolution shell). A description of the overall structure is presented, focusing on secondary structure and subunit association. The enzyme is a hexamer of alpha beta subunits. Separate alpha and beta-chains arise from an autocatalytic cleavage reaction between two serine residues, which results in the pyruvoyl cofactor. The central core of the alpha beta subunit is a beta-sandwich which consists of two face-to-face three-stranded antiparallel beta-sheets, flanked by alpha-helices on each side. The beta-sandwich creates a stable fold that allows conformational strain to be introduced across an internal cleavage region between the alpha and beta chains and places the pyruvoyl cofactor in a position for efficient electron withdrawal from the substrate. Three alpha beta subunits are related by a molecular three-fold symmetry axis to form a trimer whose interfaces have complementary surfaces and extensive molecular interactions. Each of the interfaces contains an active site and a solvent channel that leads from the active site to the exterior of the molecule. The trimers are related by a crystallographic two-fold symmetry axis to form the hexamer with an overall dumbbell shape. The interface between trimers has few molecular interactions.
Amino Acid Sequence↗
Calcium-independent subtilisin by design.
A version of subtilisin BPN' lacking the high affinity calcium site (site A) has been produced through genetic engineering methods, and its crystal structure refined at 1.8 A resolution. This protein and the corresponding version containing the calcium A site are described and compared. The deletion of residues 75-83 was made in the context of four site-specific replacements previously shown to stabilize subtilisin. The helix that in wild type is interrupted by the calcium binding loop, is continuous in the deletion mutant, with normal geometry. A few residues adjacent to the loop, principally those that were involved in calcium coordination, are repositioned and/or destabilized by the deletion. Because refolding is greatly facilitated by the absence of the Ca-loop, this protein offers a new vehicle for analysis and dissection of the folding reaction. This is among the largest internal changes to a protein to be described at atomic resolution.
Amino Acid Sequence↗
(+)-Anatoxin-a is a potent agonist at neuronal nicotinic acetylcholine receptors.
The effects of the nicotinic agonist (+)-anatoxin-a have been examined in four different preparations, representing at least two classes of neuronal nicotinic receptors. (+)-Anatoxin-a was most potent (EC50 = 48 nM) in stimulating 86Rb+ influx into M10 cells, which express the nicotinic receptor subtype comprising alpha 4 and beta 2 subunits. A presynaptic nicotinic receptor mediating acetylcholine release from hippocampal synaptosomes was similarly sensitive to (+)-anatoxin-a (EC50 = 140 nM). alpha-Bungarotoxin-sensitive neuronal nicotinic receptors, studied using patch-clamp recording techniques, required slightly higher concentrations of this alkaloid for activation: Nicotinic currents in hippocampal neurons were activated by (+)-anatoxin-a with an EC50 of 3.9 microM, whereas alpha 7 homooligomers reconstituted in Xenopus oocytes yielded an EC50 value of 0.58 microM for (+)-anatoxin-a. In these diverse preparations, (+)-anatoxin-a was between three and 50 times more potent than (-)-nicotine and approximately 20 times more potent than acetylcholine, making it the most efficacious nicotinic agonist thus far described.
Acetylcholine↗
No new state tax: universal health care coverage for children in Florida.
Florida, which this year passed a sweeping health reform measure, is considering use of a Medicaid expansion option to revamp health care delivery for its children.
Child↗
Homoanatoxin: a potent analogue of anatoxin-A.
The natural toxin anatoxin-a (AnTx) is a potent nicotinic agonist that is valuable for the study of nicotinic receptors. We have synthesized 2-(propan-1-oxo-1-yl)-9-azabicyclo[4.2.1]non-2-ene, the homologue of AnTx in which the side-chain is extended by one methylene unit from a methyl to an ethyl ketone. This chemistry would allow the generation of a tritiated product and the homologue, designated homoanatoxin (HomoAnTx), has been characterized here with that aim in mind. In competition binding assays at neuronal nicotinic ligand binding sites characterized by [3H]nicotine and [125I]-alpha bungarotoxin, HomoAnTx retained the same potency as the parent molecule, with Ki values of 7.5 nM and 1.1 microM, respectively. In contrast, it showed little inhibition of muscarinic binding defined by [3H]-quinuclidinyl benzilate. HomoAnTx is a potent nicotinic agonist in frog muscle contracture assays, having four times the potency of carbamylcholine and one tenth of the activity of AnTx itself. The N-methylated version of HomoAnTx was more than two orders of magnitude weaker in both functional and binding assays. The successful synthesis of HomoAnTx with retention of high nicotinic potency offers a route for the generation of novel, potent radiolabelled nicotinic ligands.
Animals↗
Conformational studies on (+)-anatoxin-a and derivatives.
Anatoxin-a (AnTX) is a highly potent agonist acting at the nicotinic acetylcholine receptor (nAChR) and represents a valuable tool in the study of this receptor. Molecular mechanics, semi-empirical and ab initio molecular orbital energy minimization procedures were conducted to investigate the conformation of AnTX. For each minimization procedure, the s-trans enone isomer of protonated AnTX was the energetically favoured conformer due to intramolecular electrostatic interactions. Our studies are discussed in the light of previous experimental observations and conformational studies, in addition to their importance in the development of future pharmacophore models for nAChR agonist binding.
Bacterial Toxins↗
Dipotassium and sodium/potassium crystalline picrate complexes with the crown ether 6,7,9,10,12,13,20,21,23,24,26,27-dodecahydrodibenzo[b,n]-[1,4, 7,10,13,16,19,22]octaoxacyclotetracosin (dibenzo-24-crown-8).
The crystal structures of the dipotassium and the mixed sodium/potassium picrate complexes with the crown ether dibenzo-24-crown-8 (DB24C8) were solved and found to be nearly identical. (I): NaK-pic2(DB24C8), [NaK(C6H2N3O7)2(C24H32O8)]. Mr = 966.8, triclinic, P1, a = 8.164 (2), b = 9.960 (2), c = 13.368 (3) A, alpha = 103.92 (3), beta = 108.03 (2), gamma = 93.23 (2) degrees, V = 993.0 (7) A3, Z = 1, Dm = 1.54 (T = 298 K). Dx = 1.62 (1) g cm-3, lambda = (Mo K alpha) 0.71069 A, mu = 2.37 cm-1, F(000) = 500, T = 103 K, R = 0.086 for 2904 unique reflections. (II): K2pic2(DB24C8), [K2(C6H2N3O7)2(C24H32O8)]. Mr = 982.9, triclinic, P1, a = 8.231 (4), b = 9.850 (2), c = 13.346 (4) A, alpha = 103.91 (2), beta = 106.82 (3), gamma = 93.37 (2) degrees, V = 995.7 (9) A3, Z = 1, Dm = 1.59 (T = 298 K), Dx = 1.638 (8) g cm-3, lambda (Mo K alpha) = 0.71069 A, mu = 3.30 cm-1, F(000) = 508, T = 163 K, R = 0.042 for 4835 unique reflections. Both structures feature eight-coordinated cations between alternating layers of relatively flat crown ligands and paired picrates. In the mixed-metal system the two cations are disordered between two P1-related sites; these metal sites have a coordination environment only slightly different from that in the dipotassium structure. Na+ is able to occupy an environment similar to that of K+ under the conditions of these crystals, a situation not previously observed in the chemistry of crown ethers or macrocylic multidentates.
Chelating Agents↗